![]() ![]() Inappropriate initiation of replication at late origins, because of loss of the checkpoint, probably causes the elevated level of chromatin-bound replisome proteins in the ctf18Δ mutant. We found that ctf18Δ cells are also defective in Rad53 activation, revealing that the Ctf18 complex is required for engagement of the DNA replication checkpoint. Chromatin composition abnormalities observed in ctf18Δ cells were very similar to those of an mrc1Δ mutant, which is defective in the activating the Rad53 checkpoint kinase in response to DNA replication stress. Compared with wild type, hydroxyurea-treated ctf18Δ cells exhibited increased chromatin association of replisome progression complex components including Cdc45, Ctf4, and GINS complex subunits, the polymerase processivity clamp PCNA and the single-stranded DNA-binding complex RPA. The most marked abnormalities occurred when cells were challenged with the replication inhibitor hydroxyurea. Three hundred and seven of the 491 reported chromosomal proteins were quantitated. To understand the in vivo function of the Ctf18 complex, we analyzed chromatin composition in a ctf18Δ mutant using the quantitative proteomic technique of stable isotope labeling by amino acids in cell culture. Yeast cells lacking Ctf18, the major subunit of an alternative Replication Factor C complex, have multiple problems with genome stability.
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